A major challenge today is to design an optical interrogation system that can properly interrogate a label independent detection (LID) microplate after it has been removed from and then re-inserted back into a microplate holder/XY translation stage. In particular, what is needed is an optical interrogation system that can take into account a positional misalignment of a re-positioned LID microplate so it can properly interrogate biosensors located within the wells of the re-positioned LID microplate. This need and other needs are addressed by the optical interrogation system and the method of the present invention.